Reprogramming and redifferentiation of mucosal-associated invariant T cells reveal tumor inhibitory activity

Mucosal-associated invariant T (MAIT) cells belong to a family of innate-like T cells that bridge innate and adaptive immunities. Although MAIT cells have been implicated in tumor immunity, it currently remains unclear whether they function as tumor-promoting or inhibitory cells. Therefore, we herein used induced pluripotent stem cell (iPSC) technology to investigate this issue. Murine MAIT cells were reprogrammed into iPSCs and redifferentiated towards MAIT-like cells (m-reMAIT cells). m-reMAIT cells were activated by an agonist in the presence and absence of antigen-presenting cells and MR1-tetramer, a reagent to detect MAIT cells. This activation accompanied protein tyrosine phosphorylation and the production of T helper (Th)1, Th2, and Th17 cytokines and inflammatory chemokines. Upon adoptive transfer, m-reMAIT cells migrated to different organs with maturation in mice. Furthermore, m-reMAIT cells inhibited tumor growth in the lung metastasis model and prolonged mouse survival upon tumor inoculation through the NK cell-mediated reinforcement of cytolytic activity. Collectively, the present results demonstrated the utility and role of m-reMAIT cells in tumor immunity and provide insights into the function of MAIT cells in immunity.


Introduction
Mucosal-associated invariant T (MAIT) cells belong to a family of innate-like T cells harboring semiinvariant T-cell receptors (TCRs) and recognize vitamin B2 metabolites as antigens on major histocompatibility complex class I-related gene protein (MR1) (Godfrey et al., 2019). MAIT cells play a pivotal role in immunity by bridging innate and adaptive immunities. They are abundant in humans, but rare in mice, and are associated with a number of diseases, such as bacterial and viral infections, autoimmune, inflammatory, and metabolic diseases, asthma, and cancer Godfrey et al., 2019;Toubal et al., 2019). Although previous studies implicated MAIT cells in various tumors, their role in tumor immunity remains obscure. An analysis of infiltrating immune cells revealed that the high infiltration of CD8 + CD161 + T cells, largely comprising MAIT cells, type 17 CD8 + T cells (Tc17 cells), and stem cell-like memory cells, in tumors represented a favorable prognostic signature across a wide array of human cancers (Gentles et al., 2015;Konduri et al., 2021). In mucosal-associated cancers, including lung and colon cancers, increases in MAIT cells in tumors were concomitant with a decrease in the circulation (Ling et al., 2016;Sundström et al., 2015;Won et al., 2016). While the high infiltration of MAIT cells in tumors negatively correlated with a favorable outcome in colorectal cancer (CRC) patients, the low infiltration of these cells in the tumors of hepatocellular carcinoma patients correlated with a poor prognosis, which is in contrast to the role of MAIT cells in tumor immunity (Zabijak et al., 2015;Zheng et al., 2017). Furthermore, the tumor microenvironment endowed by tumor-infiltrating lymphocytes, tumor-associated myeloid cells, and cancer-associated fibroblast plays a pivotal role in cancer progression and regression. Accordingly, tumor-residing MAIT cells exhibit an activated and/or exhausted phenotype as exemplified by the upregulation of CD39, PD-1, and CXCL13 upon the stimulation of TCR (Leng et al., 2019;Shaler et al., 2017;Yao et al., 2020). Consistent with these findings, tumor-infiltrating MAIT cells from CRC show the decreased production of cytokines that are pertinent to antitumor activity (Ling et al., 2016;Shaler et al., 2017;Sundström et al., 2015). Nevertheless, it currently remains unclear whether these changes positively or negatively affect the function of MAIT cells in tumor immunity (Cogswell et al., 2021). However, a recent study based on single-cell RNA sequencing revealed that approximately 50% of Tc17 cells in tumor-infiltrating T cells comprised MAIT cells across various tumors, and these Tc17 cells were particularly enriched in hepatocellular carcinoma and cholangiocarcinoma (Zheng et al., 2021). In addition, the frequency of Tc17 cells in melanoma patients correlated with responsiveness to anti-PD-1 therapy (Sade-Feldman et al., 2019;Zheng et al., 2021). The latter findings argue the antitumor activity of MAIT cells.
Based on these discrepancies, further studies are warranted to clarify the role of MAIT cells in tumor immunity in mice. However, difficulties are still associated with examining MAIT cells in mice for the following reasons. These cells are 10-to 100-fold less abundant in mice than in humans. Although the advent of MR1-tetramer (MR1-tet) has enabled the detection of murine MAIT cells, the paucity of these cells per se still hampers functional studies (Corbett et al., 2014;Rahimpour et al., 2015). Furthermore, there is a lack of appropriate mouse models to investigate the function of MAIT cells (Garner et al., 2018;Godfrey et al., 2019;Toubal et al., 2019). Although the development of MAIT cells is dependent on MR1, MR1-deficient mice do not necessarily reveal all aspects of MAIT cells because they are also devoid of other MR1-dependent cells and provide only limited information on immune cells that potentially interact with MAIT cells. Similarly, previous studies on MAIT cell-specific TCR transgenic mice revealed the protective roles of MAIT cells in bacterial infections, type I diabetes, and experimental autoimmune encephalitis (Chua et al., 2011;Croxford et al., 2006;Cui et al., 2015;Le Bourhis et al., 2010;Martin et al., 2009;Meierovics et al., 2013;Reantragoon et al., 2013;Sakala et al., 2015;Shalapour et al., 2012;Shimamura et al., 2011). However, the aberrant expression of transcription factors in MAIT cells impedes investigations on their precise roles in health and disease.
We herein used induced pluripotent stem cell (iPSC) technology to examine the role(s) of MAIT cells in immunity, particularly tumor immunity. We reprogrammed murine MAIT cells into iPSCs (referred to as MAIT-iPSCs), differentiated MAIT-iPSCs into MAIT-like cells (referred to as m-reMAIT cells), and adoptively transferred them into syngeneic immunocompetent mice to investigate their role. This approach not only allows us to assess the role of m-reMAIT cells as effector cells, it also provides insights into the interactions with other immune cells in tumor immunity.
In this study, we showed that 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), an agonist of MAIT cells, and 5-OP-RU-loaded murine MR1-tet (mMR1-tet) both activated m-reMAIT cells, while these reagents induced the production of a similar, but not identical, set of cytokines and chemokines in the absence of antigen-presenting cells (APCs). When adoptively transferred, m-re-MAIT cells migrated to different organs concomitant with the acquisition of maturation and prolonged mouse survival upon tumor inoculation. This tumor inhibitory activity was mediated through enhancements in the cytolytic activity of m-reMAIT cells by NK cells.
The present results imply that the adoptive transfer of m-reMAIT cells has potential as a novel tool for examining the immune functions of MAIT cells in health and disease.
Although a TCR stimulation elicits an array of signaling cascades, including protein tyrosine kinases, phosphatases, GTP-binding proteins, and adaptor proteins, we herein focused on whether 5-OP-RU activated protein tyrosine kinases and induced tyrosine phosphorylation. The results obtained showed the dose-and time-dependent phosphorylation of proteins ( Figure 1G and H). Linker for the activation of T cells (LAT) was identified as a TCR signaling component among phosphorylated proteins ( Figure 1I). Unexpectedly, the addition of mMR1-tet, a reagent widely used to detect MAIT cells, engendered similar protein tyrosine phosphorylation, as was the case for 5-OP-RU ( Figure 1G, H

and J).
These results demonstrated that MAIT-iPSCs gave rise to m-reMAIT cells upon differentiation under T-cell permissive conditions, and m-reMAIT cells were activated by 5-OP-RU and mMR1-tet in the absence of APCs concomitant with protein tyrosine phosphorylation and the production of cytokines and chemokines.

m-reMAIT cell migration into different organs
To follow the dynamics of m-reMAIT cells, cells were adoptively transferred into mice and their migration and maturation status were examined. To distinguish donor cells from endogenous MAIT cells, C57BL/6 (Ly5.1) mice received m-reMAIT cells that were Ly5.2. m-reMAIT cells and endogenous MAIT cells were detected in the thymus, bone marrow, lung, liver, spleen, intestines, and the mediastinal and inguinal lymph nodes at various endogenous MAIT cell/m-reMAIT cell ratios (Figure 2A and D). Table 1. T-cell receptor β (TCRβ) repertoires of the mouse mucosal-associated invariant T (MAIT) cell-derived induced pluripotent stem cells (iPSCs).    Since CD44 levels reflect the degree of the functional maturation of T cells (Budd et al., 1987), we examined time-dependent changes in CD44 expression in m-reMAIT cells. In comparison with endogenous MAIT cells, m-reMAIT cells showed the time-dependent upregulation of CD44 in the spleen, liver, and lungs, and lower expression in the intestines ( Figure 2B).
An analysis of other molecules relevant to the functionality of MAIT cells revealed quasi-equivalent IL-18Rα expression in the spleen, liver, and intestines to that in endogenous cells. Moreover, the expression of IL-7Rα and CXCR6 in all organs examined was lower than that in endogenous cells, while CD69 expression was quasi-equivalent to that in endogenous cells ( Figure 2C). Furthermore, the frequency of m-reMAIT cells among mMR1-tet + TCRβ + cells in primary lymphoid organs decreased, while that in peripheral tissues increased over time ( Figure 2D).
We then investigated the transcriptome in naïve m-reMAIT cells, m-reMAIT cells adoptively transferred into syngeneic mice, and endogenous MAIT cells from the recipient mice to follow the evolution of transcripts by RNA sequencing. A principal component analysis (PCA) revealed that the transcriptome of naïve m-reMAIT cells clustered upon adoptive transfer, which was similar to that of endogenous MAIT cells from lamina propria lymphocytes (Figure 2-figure supplement 1). We then focused on the transcripts relevant to the identity and function of MAIT cells Salou et al., 2019). We found that the expression of transcripts, such as Il7r, Il12rb,Il18r,Il18rap,Prf,Gzmb,Tnf,Ifng,Il22,Slamf7, Abcb1a (encoding the efflux pump), Zbtb16 (encoding PLZF), Tbx21 (encoding T-bet), and Zfp683 (encoding Hobbit), was upregulated upon the adoptive transfer of naïve m-re-MAIT cells, and the expression levels of some transcripts reached those in endogenous MAIT cells (Figure 2-figure supplement 2). Similar results were obtained for transcripts pertinent to the tissue repairing and tissue residency signature (Figure 2-figure supplements 3 and 4; Leng et al., 2019;Salou et al., 2019;Yanai et al., 2016). Transcripts relevant to the V-D-J recombination machinery, such as Rag1, Rag2, Hmgb1, Dntt, and Lig4, were silenced across the organs upon adoptive transfer ( Figure 2-figure supplement 5).
(C) MR1-dependent activation of m-reMAIT cells. The percentage of CD69 + cells among m-reMAIT cells cultured with CH27/mMR1, challenged as in (B) in the presence of the anti-MR1 antibody (■) or the isotype control antibody (◯). (D) 5-OP-RU dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with various concentrations of 5-OP-RU. Representative data from two independent experiments are shown. (E) mMR1-tet dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with the indicated amounts of mMR1tet. Representative data from two independent experiments are shown. (F) 5-OP-RU-and mMR1-tet-induced cytokines and chemokines. m-reMAIT cells were stimulated with various concentrations of 5-OP-RU (◯) or mMR1-tet (■) and the resultant cytokines and chemokines were quantified with LegendPlex. The concentrations at which each reagent induced a similar degree of activation (% CD69) are shown as relative concentrations (0.1-100 nM for 5-OP-RU and 0.01-10 μg/ml for mMR1-tet). The number on the X-axis corresponds to that in (        These results indicated that m-reMAIT cells migrated into different organs accompanying maturation upon adoptive transfer in immunocompetent mice.

Tumor inhibitory activity of m-reMAIT cells
Since the role of MAIT cells in tumors has been a target for intensive scrutiny, we attempted to clarify whether m-reMAIT cells interfered with tumor metastasis and prolonged mouse survival Toubal et al., 2019). A previous study on B16F10 melanoma suggested that MAIT cells induce tumor development in a manner that is dependent on MR1 and agonists, such as 5-OP-RU (Yan et al., 2020). While B16F10 significantly upregulated MR1 on the cell surface with the 5-OP-RU challenge, a similar change was not observed for Lewis lung carcinoma (LLC) ( Figure 3A). Therefore, the use of LLC allows for assessments of the role of m-reMAIT cells in tumor immunosurveillance independent of the 5-OP-RU-MR1 axis.
The adoptive transfer of m-reMAIT cells into mice followed by the LLC inoculation prolonged survival in a dose-dependent manner. While 3 × 10 5 m-reMAIT cells slightly prolonged survival over the control, more than 1 × 10 6 m-reMAIT cells significantly prolonged survival ( Figure 3B). Nevertheless, multiple transfers of m-reMAIT cells failed to significantly prolong survival ( Figure 3C). In contrast to these results, the adoptive transfer of m-reMAIT cells did not suppress tumor growth at any dose following the subcutaneous inoculation of LLC, which represents an in situ tumor growth model ( Figure  These results implied that m-reMAIT cells inhibited tumor metastasis rather than suppressing tumor growth in situ.

Cytolytic activity of m-reMAIT cells in combination with NK cells
While the above results indicated that m-reMAIT cells functioned to suppress metastasis, it currently remains unclear whether other immune cells are involved in this tumor inhibitory activity. NK cells are an essential innate sentinel in tumor immunosurveillance, and previous studies reported contradicting findings regarding the role of MAIT cells on NK cells in tumor immunity. Yan et al. demonstrated that MAIT cells promoted tumor growth by suppressing the activity of NK cells and T cells (Yan et al., 2020), while others suggested that the activation of MAIT cells in vivo strengthened antitumor activity concomitant with an enhanced NK cell response (Petley et al., 2021). Therefore, we investigated whether and how m-reMAIT cells and NK cells mutually affect their functions in tumor immunity. Interactions between NK cells and m-reMAIT cells were assessed in a coculture. While m-reMAIT cells were activated in an NK cell dose-dependent manner, the activation of NK cells was less prominent ( Figure 4A). Moreover, the coculture led to the production of Th1 and Th17 cytokines as well as inflammatory chemokines in an NK cell dose-dependent manner ( Figure 4B).
To obtain further insights into the molecular events underlying this interaction, NK cells and m-re-MAIT cells were purified with FACS after the coculture and each subset was examined for transcripts relevant to cytolytic function ( Figure 4C and D). Ifng was induced in m-reMAIT cells, but not in NK The online version of this article includes the following source data and figure supplement(s) for figure 2:          cells. Similarly, the coculture enhanced the transcripts for serine proteases, such as Grza, Grzb, and Tnf, in m-reMAIT cells and NK cells (Trapani and Smyth, 2002). In contrast, the expression of the transcripts for the TNF superfamily, including Fasl and Tnsf10, was only stimulated in m-reMAIT cells. Moreover, the expression of Ccl4 (Mip1b), Ccl5 (Rantes), Il17a, and Il6 was upregulated in m-reMAIT cells in contrast to only Ccl5 (Rantes) in NK cells.
We then investigated whether this change had an impact on CD69 and CD107a, a marker of cytolytic granule exocytosis. While the coculture with m-reMAIT cells did not markedly upregulate the expression of CD69 (CD69 + cells) in NK cells, the addition of Yac-1, an NK cell-sensitive tumor cell line (alone or in combination with m-reMAIT cells), increased the percentage of CD69 + cells. However, these combinations resulted in negligible changes in the percentage of CD69 + CD107a + cells     Figure 4E, upper panels). In contrast, incubation with NK cells or Yac-1 enhanced the percentage of CD69 + cells in m-reMAIT cells. Moreover, a coculture with NK cells together with Yac-1 resulted in further increases, and similar results were obtained for CD69 + CD107a + cells in m-reMAIT cells ( Figure 4E, lower panels). We then examined whether the above activation and/or expression of CD107a reflected lytic activity against tumor cells. m-reMAIT cells lysed Yac-1, an NK cell-sensitive tumor cell line, as efficaciously as NK cells, and these cells synergistically enhanced killing ( Figure 4F). We extended our study to LLC, an NK cell-insensitive cell line. NK cells alone did not induce cell death in LLC, whereas m-reMAIT cells did. Moreover, the combination of these cells exhibited synergistically enhanced lytic activity ( Figure 4G).
We then investigated the significance of the interaction between NK cells and m-reMAITs cell in vivo. To assess the role of NK cells in m-reMAIT cell-mediated anti-metastasis activity, NK cells were depleted in mice with an anti-Asialo GM1 antibody (AsG). This depletion did not significantly affect the survival of control mice. However, the AsG treatment abrogated the survival advantage of m-re-MAIT cells, highlighting the stimulatory role of NK cells in m-reMAIT cell-mediated tumor inhibitory activity ( Figure 4H).
These results demonstrated that the intrinsic cytolytic activity of m-reMAIT cells against tumors was bolstered by NK cells concomitant with the upregulation of the relevant arsenals.

Discussion
The present results revealed that m-reMAIT cells were competent in TCR signaling. The activation of m-reMAIT cells and subsequent production of cytokines and chemokines in the absence of APCs suggested that 5-OP-RU directly operated via MR1 in m-reMAIT cells ( Figure 1D-F). 5-OP-RU may be passively transported into cells, which in turn promotes the release of antigen-loaded MR1 from the endoplasmic reticulum (ER) onto the plasma membrane, thereby enabling signaling via TCR in m-reMAIT cells (referred to as a cis-acting signal) regardless of the apparent absence of MR1 on the cell surface ( Figure 1B and D, Figure 1-figure supplement 1I; McWilliam et al., 2016). In contrast, mMR1-tet directly binds to TCR on m-reMAIT cells and elicits signals (referred to as trans-acting independent experiments are shown. (C) Transcripts relevant to cytolytic activity in m-reMAIT cells. Ifng, Gzma,Gzmb,Tbf,Gzmk,Pfr1,Fasl,Tnfsf10,Il6,Il17a,Il22,Ccl3,Ccl4,Ccl5, and Ccl22 in m-reMAIT cells cultured with NK cells were quantified with qRT-PCR. m-reMAIT cells and NK cells were sort-purified after the coculture (purity >98%) or cultured individually. The expression of each transcript was normalized with Gapdh, and fold changes in the relative expression of the transcript in m-reMAIT cells cultured with NK cells relative to that in m-reMAIT cells cultured alone are shown. Data are representative of three independent experiments. (D) Transcripts relevant to cytolytic activity in NK cells. Fold changes in the relative expression of the indicated transcript as described in (C) in NK cells cultured with m-reMAIT cells relative to that in NK cells alone are shown. Representative data from three independent experiments are shown. (E) Activation and degranulation of NK cells and m-reMAIT cells. The expression of CD69, an activation marker, and CD107a, a marker for the exocytosis of cytolytic granules, was assessed under various culture conditions. The percentages of CD69 + cells and CD69 + CD107a + cells among NK cells alone (control), NK cells cocultured with m-reMAIT cells (+reMAIT), NK cells cultured with Yac-1 (+Yac-1), and NK cells cocultured with m-reMAIT cells and Yac-1 (+reMAIT/Yac-1) (upper panels). The percentages of CD69 + cells and CD69 + CD107a + cells among m-reMAIT cells alone (control), m-reMAIT cells cocultured with NK cells (+NK), m-reMAIT cells cocultured with Yac-1 (+Yac-1), and m-reMAIT cells cocultured with NK cells and Yac-1 (+NK/Yac-1) (lower panels). Data are representative of three independent experiments. (F) Cytolytic activity against Yac-1. Cytolytic activity of m-reMAIT cells (reMAIT ◯), NK cells (NK □), and NK cells plus m-reMAIT cells (NK+reMAIT •). Cytolytic activities (% lysis) at different effector (NK cells, m-reMAIT cells, and NK cell+m-reMAIT cells)/Target (Yac-1) (E/T) ratios are shown. Representative data from three experiments are shown. The significance of differences between the groups at the indicated E/T ratio assessed with a two-way ANOVA is shown (*p<0.05, **p<0.01, ***p<0.005). From the top, NK+reMAIT vs. reMAIT, NK+reMAIT vs. NK, and reMAIT vs. NK. Data are representative of three independent experiments. (G) Cytolytic activity against Lewis lung carcinoma (LLC). The cytolytic activities of m-reMAIT cells (◯), NK cells (□), and m-reMAIT cells plus NK cells (•) against LLC at the indicated E/T ratio are shown as % lysis. The significance of differences between the groups is calculated as in (E). Data are representative of three independent experiments. (H) NK cell-dependent extension of survival. C57BL/6 mice were divided into two groups, one that received 1 × 10 6 m-reMAIT cells (reMAIT) and another that was left untreated (n). Each group was further divided into two subgroups, one that received consecutive injections of anti-Asialo GM1 (AsG) (-1 and 16 days, 50 μg/mouse) after the LLC inoculation (3 × 10 5 i.v.) and another that was left untreated (n). Survival was monitored thereafter. Representative data from two independent experiments are shown (n = 10-14/ group). p-Values between the indicated groups are shown (the log-rank test).
The online version of this article includes the following source data for figure 4: Source data 1. Antitumor activity of m-reMAIT cells bolstered by NK cells.  . Therefore, 5-OP-RU and mMR1-tet may have elicited similar signals through serine/threonine and/or tyrosine phosphorylation or dephosphorylation via TCR, which ultimately resulted in a different profile of cytokine and chemokine production ( Figure 1F-H and J; Gaud et al., 2018;Nausch and Cerwenka, 2008). In this respect, the signaling pathway(s) responsible for the production of IL-2, IL-4, MIP1α CCL3 (MIP-1α), and CCL4 (MIP-1β) may be common between 5-OP-RU and mMR1-tet. In contrast, mMR1-tet may have activated the signaling pathways pertinent to the activation of Th17 as evidenced by the production of Th17 cytokines, such as IL-17F, IL-22, and IL-23, concomitant with inflammatory chemokines, including CCL5, CCL2, CXCL1, and CXCL6, in addition to TNF-α ( Figure 1F). These results suggest that mMR1-tet signals, in part, through NF-κB, while 5-OP-RU does not .
While 5-OP-RU induced protein tyrosine phosphorylation, the identity of these proteins remains unclear, except for LAT ( Figure 1G and H). Since LAT forms a hub that interacts with many signaling molecules, such as transmembrane receptors, phosphatases, kinases, guanine nucleotide exchange factors, GTP-activating proteins, ion channels, transporters, and ubiquitin ligases, further studies will provide insights into the proteins responsible for MAIT-TCR signaling and reveal the differences in signaling between conventional T cells and MAIT cells (Malissen et al., 2014).
It is important to note that the challenge with mMR1-tet induced the activation of m-reMAIT cells and production of cytokines and chemokines. This suggests that MAIT cells prepared from animals, including humans and mice, are unintentionally stimulated during experiments. Therefore, the interpretation of data requires caution ( Figure 1E, F and J).
Although a previous study suggested that MAIT cells comprise MAIT1 and MAIT17 subsets characterized by the Th1 and Th17 transcriptomes, respectively, the present results revealed that m-reMAIT cells produced Th1, Th2, and Th17 cytokines (Salou et al., 2019). This may reflect a developmental stage at which m-reMAIT cells are en route to functional maturation and m-reMAIT cells generated in vitro represent the most immature stage. Therefore, it is tempting to postulate that the interaction between nascent MAIT cells and double-positive thymocytes favors the differentiation of naïve MAIT cells into MAIT1 and MAIT17 in vivo. The results on the adoptive transfer of m-reMAIT cells indicated that nascent MAIT cells egressed from the thymus migrate into any organs concomitant with the upregulation of CD44, a cell adhesion receptor, memory, and/or activation marker ( Figure 2B; Benlagha et al., 2005). It is of interest that the expression of IL-7Rα in m-reMAIT cells became quasiequivalent to that in endogenous cells in the intestines. Since IL-7 from intestinal epithelial cells plays a pivotal role in their homeostasis, the upregulation of IL-7Rα in m-reMAIT cells may mirror an intrinsic and primary role in this homeostasis and/or in the integrity of the intestinal epithelial barrier (Rouxel et al., 2017;Shalapour et al., 2012). Alternatively, it may reflect the role of MAIT cells in IL-17A and Th1 cytokine production, which is dependent on IL-7 (Tang et al., 2013). Taken together with the time-dependent upregulation of CD44, increases in IL-18Rα and CXCR6 across organs indicated that m-reMAIT cells matured in the host. Thus, naïve m-reMAIT cells, defined as mMR1-tet + TCRβ + cells generated in vitro, may be promoted maturation in terms of transcriptional program upon adoptive transfer into the recipient mice, as evidenced by the acquisition of cytokine receptors, the effector molecules involved in cytotoxicity and tissue repairing, and those relevant to tissue residency (Figure 2-figure supplements 2-4). Moreover, the silencing of transcripts relevant to V-D-J recombination may ensure the continuum of the identity of m-reMAIT cells in the recipient (Figure 2-figure  supplement 5). These results have important implications for future cell therapy with human iPSCderived MAIT cells. Further studies are needed to clarify whether and how the adoptive transfer of naïve m-reMAIT cells affects epigenetic states, and whether this change leads to the generation of bona fide MAIT cells.
m-reMAIT cell dose-dependent mouse survival indicated their tumor inhibitory activity and function as effector cells in tumor surveillance ( Figure 3B). However, since three consecutive injections of m-reMAIT cells failed to confer survival superiority, the niche for MAIT cells appeared to be saturated. Therefore, further studies are warranted to investigate whether a lymphopenia-inducing regimen, such as irradiation and/or cyclophosphamide, confers a difference in survival.
The failure of m-reMAIT cells to inhibit tumor growth in situ regardless of their mobilization to the skin indicated their poor infiltration into tumors rather than the loss of antitumor activity (Figure 3figure supplement 1). Therefore, future research that focuses on whether an intratumor injection of m-reMAIT cells inhibits tumor growth is needed.
While the importance of NK cells in tumor surveillance is well known, that of MAIT cells remains elusive (Cogswell et al., 2021;Guillerey et al., 2016;Sharma et al., 2017). The present results showing the NK cell-dependent activation of m-reMAIT cells and the subsequent production of inflammatory cytokines and chemokines suggested that NK cells stimulated the tumor inhibitory effects of MAIT cells ( Figure 4A and B) Figure 4C-G). The upregulation of Grzb, Fasl, and Tnfsf10 in m-reMAIT cells suggested that cytolytic activity comprised exocytosis-as well as caspasemediated killing (Rossin et al., 2019;Trapani and Smyth, 2002). NK cell depletion compromised the survival of mice, which is consistent with the above findings, further highlighting the role of NK cells in boosting the tumor inhibitory activity of m-reMAIT cells in vivo. While NK cells recognize NKG2D ligands and/or the lack of MHC I on tumor cells, the molecular mechanisms by which m-reMAIT cells recognize and eliminate tumor cells warrant further study (Nausch and Cerwenka, 2008).

. Moreover, the enhanced expression of transcripts relevant to the cytolytic function of m-reMAIT cells upon an interaction with NK cells in vitro indicated that m-reMAIT cells exhibited cytolytic activity in vivo (
Contrary to our results, MAIT cells have been shown to induce the metastasis of melanoma B16F10 (Yan et al., 2020). Although the reason for this discrepancy currently remains unclear, differences in the dynamics of MR1 shuttling between ER and the plasma membrane upon a 5-OP-RU challenge or putative ligand(s) present in the tumor milieu (TM) may be a key feature. Since LLC did not strongly upregulate MR1 on the cell surface when stimulated ( Figure 4A), 5-OP-RU may have bolstered B16F10 tumorigenicity through MR1. The MR1-elicited signal may suppress the function of NK cells for B16F10, whereas that from putative ligand(s) present in TM via MR1 did not, thereby enhancing or preserving NK cell functions. Further studies are needed to elucidate the underlying mechanisms, which will provide a novel avenue for tumor immunotherapy with iPSC-derived MAIT cells.
Although we showed that m-reMAIT cells exhibited cytolytic activity against LLC together with NK cells in vitro (Figure 4C and E-G), difficulties are associated with demonstrating that m-reMAIT cells isolated from mice exhibit similar lytic activity ex vivo. This is due to the low recovery of m-reMAIT cells and the compulsory use of mMR1-tet in cell preparations, which may interfere with cytolytic activity. Furthermore, it currently remains unclear whether these results are applicable to other cancer cells, such as B16F10, if the use of 5-OP-RU enhances or inhibits the cytolytic activity of m-reMAIT cells. Furthermore, since the transcriptome in m-reMAIT cells upon adoptive transfer still differed from that in endogenous cells, caution is needed when extrapolating the present results to MAIT cells in vivo.
In summary, the adoptive transfer model with m-reMAIT cells used in this study opens a new avenue for exploiting the function of MAIT cells and providing insights into their interaction(s) with immune cells in immunity.

Mice
All mouse experiments were performed with approval from the Institutional Animal Care and Use Committee of Dokkyo Medical University (permit number 1215). C57BL/6NJcl mice were purchased from CLEA Japan (Tokyo, Japan). C57BL/6 (Ly5.1) mice were obtained from the RIKEN Bioresource Center and bred in-house. All mice were housed in the Animal Research Center, Dokkyo Medical University, under specific pathogen-free conditions with controlled lighting and temperature with food and water provided ad libitum. Male C57BL/6NJcl mice aged 6 weeks were used to isolate MAIT cells for the generation of iPSCs. Male and female mice aged between 8 and 12 weeks were used in adoptive transfer experiments and tumor experiments.

Cell lines
The cell lines used in this study were obtained as described in the Key resources table. These cells were periodically checked for mycoplasma contamination with mycoplasma detection kit (Takara Bio, Japan). Mycoplasma was not detected throughout the experiments. OP9/DLL1 cells were maintained in αMEM supplemented with 20% FBS. The mouse cancer cell lines B16F10, CH27, CH27/mMR1, EL4, and LLC were cultured in DMEM supplemented with 10% FBS, while RL-♂1, WT3, WT3/mMR1, and Yac-1 were cultured in RPMI 1640 supplemented with 10% FBS at 37°C in 5% CO 2 .

Antibodies
The antibodies used in this study are listed in the Key resources table.

Oligonucleotides
The oligonucleotides used in this study are summarized in the Key resources table.

Preparation of mouse immune cells Spleen, thymus, and lymph nodes
Tissues were prepared by mashing through a 40 µm mesh cell strainer with a syringe plunger. Single cells were suspended in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES pH 7.0, 0.1 mM 2-mercaptoethanol, and 100 IU/ml of penicillin/streptomycin (referred to as cR10) and spun down at 400 × g for 4 min. To lyse red blood cells, the cell pellet was suspended in autoclaved ice-cold MilliQ water for 15 s and immediately neutralized with 4% FBS in 2× PBS. After centrifugation, cells were resuspended in cR10.

Lungs and liver
Single-cell suspensions from the lungs and liver were prepared using enzymatic digestion. Briefly, tissues were placed into a GentleMACS C-tube (Miltenyi Biotec) and cut into approximately 5 mm 3 pieces. 4 ml of tissue digestion solution (90 U/ml collagenase Yakult, 275 U/ml collagenase type II, 145 PU/ml Dispase II, and 4% BSA in HBSS) was added per tissue, and tissues were homogenized using the GentleMACS dissociator (Miltenyi Biotec) with the following program: m_lung_01_02 for the lungs and m_liver_03_01 for the liver. Suspensions were then incubated at 37°C for 30 min under gentle rotation, followed by dissociation with m_lung_02_01 for the lungs and m_liver_04_01 for the liver, and subjected to discontinuous density centrifugation over layers of 40 and 60% Percoll at 400 × g for 20 min. Cells were recovered from the 40-60% Percoll interface, washed with PBS, and then suspended in cR10.

Intestines
The intestines were longitudinally incised, and their contents were thoroughly washed out three times by vigorous shaking. Tissues dissected into 1 cm pieces were placed into a 50 ml conical tube and washed vigorously by shaking three times with PBS. After discarding the supernatant, the tissues were treated with 40 ml of intraepithelial lymphocyte-washing solution (HBSS containing 1 mM DTT, 5 mM EDTA, and 1% BSA) by shaking vigorously at 37°C for 30 min under gentle rotation. After washing three times with MACS buffer (PBS containing 2 mM EDTA and 0.5% BSA), the tissues were washed again with 40 ml HBSS. After removal of the supernatant, the tissues were placed into a GentleMACS C-tube (Miltenyi Biotec), cut into small pieces with scissors, and 4 ml of the tissue digestion solution (see above) was added. The tissues were processed using the program m_brain_01_02 and digested at 37°C for 30 min under gentle rotation followed by dissociation with the program m_intestine_01_01. Cell suspensions were subjected to Percoll discontinuous density centrifugation, and isolated cells were resuspended in cR10.

PCR detection of the rearranged configuration of TCR loci in MAIT-iPSCs
To confirm that MAIT-iPSCs stemmed from MAIT cells, PCR detecting the rearranged configuration of Trav specific for MAIT cells was performed with the primer sets ADV19 and AJ33. Genomic DNA was prepared from MAIT-iPSCs with NaOH. To detect Trbv in MAIT-iPSCs, total RNA was prepared from m-reMAIT cells (days 20-28 of differentiation, varying according to the clones) using the RNeasy Mini kit (QIAGEN). cDNA was synthesized with the first-strand cDNA synthesis kit (Thermo Fisher Scientific) and subjected to PCR with the primer sets TRBV13 and TRBC-Rev, and TRBV19 and TRBC-Rev followed by DNA sequencing (Fasmac). The usage of TRBV-D-J was analyzed using IGBLAST (NCBI, NIH https://www.ncbi.nlm.nih.gov/igblast/).

Preparation of 5-OP-RU
An aliquot of 5-A-RU (3.6 mM in DMSO) was incubated with three volumes of 1 mM methylglyoxal (MilliQ water) at 37°C for 30 min and used for assays. The appropriate synthesis of 5-OP-RU was confirmed with LC-MS/MS (Triple Quad 5500, Sciex). m-reMAIT cell activation assay m-reMAIT cells (L3-1, L7-1(L7), L11-1, L15-1, and L19-1) were incubated with the indicated concentration of 5-OP-RU or mMR1-tet at 37°C in 5% CO 2 for 18 hr in the presence or absence of equal numbers of CH27, CH27/mMR1, WT3, and WT3/mMR1, and then subjected to a CD69 expression analysis by flow cytometry. The culture supernatant was used for cytokine and chemokine quantification (see below). To examine whether the above activation of m-reMAIT cells was dependent on MR1, an anti-MR1 antibody (26.5) or isotype antibody (10 μg/ml) was added 1 hr prior to the addition of 5-OP-RU.

Doubling time estimation with CFSD-SE (CFSE)
m-reMAIT cells (differentiation on days 18-23) were labeled with CFSE (1.0 μM) and cultured on OP9/ DLL1 in αMEM containing 20% FBS (BioSera) and 1 ng/ml mouse IL-7. CFSE intensity in the cells was monitored daily by flow cytometry with 488 nm excitation and a bandpass filter of 530/30 nm. The proliferation rate of m-reMAIT cells was calculated from a logarithmical growth curve based on the intensity of CFSE.

Flow cytometry
Cells were stained with the antibodies listed in the Key sources table. 7-AAD or Zombie Violet (BioLegend) was used to discriminate between live/dead cells. To stain the transcription factors PLZF and RORγt, cells stained with the surface markers were fixed and permeabilized with the Transcription Factor Buffer Set (BD Biosciences), and then stained with transcription factor antibodies. In CD107a staining, a fluorochrome-labeled CD107a antibody was added to the culture prior to the assay. Cells were analyzed with the MACSQuant cell analyzer (3 lasers, 10 parameters, Miltenyi Biotec) or the AttuneNxT acoustic focusing cytometer (4 lasers, 14 parameters, Thermo Fisher Scientific). Data were processed using FlowJo software (version 9 or 10, BD Biosciences). Cell sorting was performed using a FACSJazz cell sorter (two lasers, eight parameters, BD Biosciences).

Cytokine and chemokine quantification
The quantification of cytokines and chemokines was performed with the LegendPlex mouse Th cytokine panel, mouse cytokine panel 2, and mouse proinflammatory chemokine panel according to the protocol provided by the manufacturer (BioLegend).

Transcriptome analysis
FPKM was calculated from the RNA-seq read count data (121,806 genes) and logarithmically converted and normalized based on the expression level of the housekeeping genes with RUV function in R package (Jacob et al., 2016). Genes exhibiting a constant expression level across the samples were considered to be housekeeping (108 genes). PCA was calculated with 'prcomp' and visualized with 'ggplot2' in R package using the above data. Then the genes relevant to MAIT cell identity, function, and development were categorized, and the heatmap was created with 'pheatmap' in R package. Only differentially expressed genes (naïve m-reMAIT cell/endogenous MAIT cells or adoptively transferred m-reMAIT cells < 0.5 or > 2) are shown. Differentially expressed genes were analyzed with 'TCC' in R package (Kadota et al., 2012;McCarthy et al., 2012;Sun et al., 2013).
Time course of MR1 expression in cancer cell lines upon the 5-OP-RU challenge B16F10, EL4, LLC, RL♂1, and Yac-1 were incubated with 600 nM of 5-OP-RU. Thereafter, the geometric mean fluorescent intensity (gMFI) of MR1 on the cell surface was followed by flow cytometry. gMFI at the indicated time point relative to that at the nontreated state (time 0) was calculated and shown as relative gMFI.
Tumor studies LLC suspended in HBSS was intravenously (i.v.) inoculated (3.0 × 10 5 cells/mouse) 6 days after the adoptive transfer of m-reMAIT cells (1.0 × 10 6 cells/mouse unless otherwise indicated, i.p.) or HBSS alone. In the survival assay, mice were considered to be dead when they showed a humane end point, such as acute weight loss, hypothermia, and severe gait and/or consciousness disturbance, and the survival time of mice was plotted as a Kaplan-Meier curve. In the in situ tumor growth analysis, LLCs (3.0 × 10 5 cells/mouse) were subcutaneously inoculated 6-8 days after the adoptive transfer of m-reMAIT cells (0.3 × 10 6 , 1.0 × 10 6 , and 3.0 × 10 6 cells/mouse, i.p.) or HBSS alone. Tumor sizes were measured with calipers every 3 days. Tumor volumes were calculated using the following formula: v = (tumor width) 2 × (tumor length)/2.

Interaction between NK cells and m-reMAIT cells
NK cells were isolated from C57BL/6 mice (male and female) with the MojoSort mouse NK cell isolation kit (BioLegend) (purity >85% based on the flow cytometric analysis with NK1.1 and CD49b antibodies) and cocultured with m-reMAIT cells at 37°C in 5% CO 2 for 18 hr at the indicated ratio. In parallel, m-reMAIT cells and NK cells were cultured individually under the same conditions, and CD69 expression in NK cells (gated as NK1.1 + CD49b + cells) and m-reMAIT cells (gated as TCRβ + mMR1-tet + cells) was analyzed using flow cytometry. Cytokines and chemokines released in the culture were measured with LegendPlex at the indicated ratio. In CD69 and CD107a expression analyses, the above columnisolated NK cells (1 × 10 5 cells) and m-reMAIT cells (1 × 10 5 cells, purity >95%) were cultured individually or cocultured in the absence or presence of the same number of Yac-1 at 37°C in 5% CO 2 for 18 hr. The percentages of CD69 + cells and CD69 + CD107a + cells among NK cells and m-reMAIT cells were then measured using the MACSQuant flow cytometer.

Semi-quantitative PCR
NK cells cocultured with m-reMAIT cells (NK cells/m-reMAIT cells ratio = 1) were sort-purified as NK1.1 + CD49b + NK cells and TCRβ + mMR1-tet + m-reMAIT cells, respectively. RNA from each subpopulation was extracted with the RNeasy Mini kit (QIAGEN), and cDNA was synthesized with the firststrand cDNA synthesis kit (Thermo Fisher Scientific) and then subjected to semi-quantitative PCR. PCR was performed with CYBR Green reagent (Nippon Genetics) using the following program: at 95°C for 5 min (90°C for 15 s, 60°C for 60 s) × 50 cycles (Light/Cycler Nano, Roche). The primer sets used in this study are described in the Key resources table.

Cytolytic activity
The cytolytic activities of NK cells and m-reMAIT cells were measured with Yac-1 and LLC as target cells. Yac-1 cells (1.0 × 10 4 cells/assay) were labeled with CFSE 24 hr prior to the addition of effector cells, while LLC cells (1.0 × 10 4 cells/assay) were not labeled. Target cells were then incubated with the effector cells at the indicated E/T ratio at 37°C in 5% CO 2 for 4 hr and 16 hr for Yac-1 cells and LLC cells, respectively. E/T ratios of 1/1, 1/3, and 1/10 in the assay using the combination of NK cells and m-reMAIT cells contained twice the number of effector cells than NK cells or m-reMAIT cells alone. Dying target cells were stained with the Zombie Flexible Viability kit according to the manufacturer's instructions. Percent lysis was calculated by the formula {(% of Zombie + cells among target cells in the presence of effector cells) − (% of Zombie + cells among target cells in the absence of effector cells)}/ 100 − (% of Zombie + cells among target cells in the absence of effector cells)/100.

Quantification and statistical analysis
Statistical analyses were conducted using Prism 9 for macOS (GraphPad). The log-rank test was used for survival analyses between the two indicated groups. A two-way ANOVA was employed to assess the significance of differences among the various effector cells (NK cell, m-reMAIT cells, and NK cells plus m-reMAIT cells) in lysis assays.